The present invention relates to liquid flow photometers for fluorescence light and/or scattered light measurement, having a measuring area where particles flow through.
Liquid flow photometry is a measuring technique increasingly more often applied within several fields of cell biology, medicine and physical-chemical measurements of particles of almost any material. In a liquid flow photometer photometric signals from single particles of microscopic size are measured when they--carried by a laminar liquid flow--pass one by one through a focus of excitation light of high intensity. The particles are centered in the liquid flow by so called hydrodynamic focussing. The types of photometric signals one is able to register, are: (1) the particles' fluorescence, (2) the light scattering caused by the particles, and (3) light absorption caused by the particles. The two first of these signal types have proved to be particularly informative.
As a typical example of the application of liquid flow photometry one can mention the measuring of the content of various components, as for instance DNA or protein, in single cells. For this purpose the cells are stained with a fluorescent dye binding specifically and quantitatively to the component in question. In this way every cell passing through the photometer's excitation focus will cause a pulse of fluorescence light. This light is picked up by a light detector. The signal from this--which is proportional to the cell's content of dye and thus of the cell component in question--is measured and stored in an electronic memory according to its size--a so called multichannel pulse height analyser. In this way several thousand cells can be measured per second with very great accuracy, and a histogram of the number of cells as a function of the cell's content of the cell component in question is obtained. The excitation light scattered by the cell as it passes the photometer's focus can be registered by another detector. Depending on the scattering angle this signal gives information on the cell's cross section or volume, and also on its inner structure and density.
In most types of liquid flow photometers the excitation light is induced by a laser. The liquid flow carrying the particles passes through the laser beam. The particles' fluorescence and light scattering are registered by two separate optical systems located beside the laser beam. In certain types of such liquid flow photometers the liquid flow is a free jet in the air. In others the liquid flow passes through a closed chamber.
The types of liquid flow photometers mentioned above are complicatedly built, and this makes them very expensive and difficult to operate. A new type of liquid flow system, which can be applied as an accessory to a standard fluorescence microscope has been described in the Norwegian Patent Application No. 791229 Fitted with a suitable light detector the microscope is converted to a liquid flow photometer which,--as regards fluorescence detection--has proved to be up to the level of the far more expensive laser-based instruments, while it is also very easy to operate. As described in the above mentioned patent application, in this liquid flow system a hydrodynamically focussed flow of particles in a laminar liquid jet is induced by a nozzle. The nozzle is placed so that the liquid flow falls at an oblique angle onto a cover glass and is thus converted to a flat, laminar flow moving along the surface of the cover glass. Because they are centered in the liquid jet, the particles will flow across the glass one by one within a narrow sector. The cover glass is placed so in relation to the microscope's objective, that the particle flow on the glass passes through the objective's focus, i.e. the microscope's measuring area.
The above-described microscope has Epi-illumination, which means that the excitation light is focussed onto the measuring area by the same objective as that collecting the fluorescence light. The objective is preferably of the oil immersion type to obtain maximum numerical aperture and correspondingly high sensitivity.
The liquid flow photometer described in the above-mentioned patent application can only measure the particles' fluorescence. Another of the instrument's limitations is that the source of the excitation light is a conventional high pressure Hg- or Xe-lamp. The light intensity from such lamps may vary somewhat, partly because the lamp's arc is not completely stable, and partly because the light emission from the lamp generally decreases with the lamp's utility time. These variations reduce reproducibility of the measurements.